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We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin. One plasmid contained a single complete copy of the rRNA gene and its flanking sequences, while the other had an additional rDNA origin of replication. In all B or rmm1 Tetrahymena cell lines transformed with the plasmids, rDNA sequences from the plasmid were found in palindromic rDNA molecules. In one transformant line, a small amount of the plasmid was also retained in a form with the original circular restriction map. Our results show that the plasmids underwent homologous recombination with one arm of the endogenous rDNA to give heteropalindromic rDNA, or with both arms of the palindrome to form homopalindromic rDNA. The resulting recombinant molecules were able to replace the recipient's original rDNA completely, providing strong evidence that C3 rDNA sequences in the donor DNAs confer a replication advantage over recipient rDNA. Thus microinjection of circular plasmids provides a method for replacement of an endogenous gene or gene fragment with exogenous sequences.

A telomere terminal transferase activity was identified in developing macronuclear extracts from Euplotes crassus. The activity was essentially unregulated in vitro: up to 50 tandem repeats of the Euplotes telomeric repeat sequence TTTTGGGG were added onto synthetic telomeric oligonucleotide primers. Both the structure of the telomere substrate and its 3'-terminal sequence were recognized. The activity was destroyed by low concentrations of RNase A.

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5' end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.

A circular plasmid containing a complete Tetrahymena thermophila rRNA gene (rDNA), with a tandem repeat of a 1.9-kilobase-pair segment encompassing the replication origin and the rRNA promoter, and a polylinker in the 3' nontranscribed spacer, was used to transform T. thermophila by microinjection. Most (20/21) stable transformants contained only recombinant linear palindromic rDNA molecules carrying rDNA sequences from both the donor plasmid and the recipient cell, as shown previously. However, in one transformant, the circular plasmid initially outreplicated the endogenous rDNA and was the major rDNA form for up to 65 generations. Stable circular replicons have not been reported previously in Tetrahymena. A single point mutation (+G) was identified in the repeated promoter of the plasmid maintained in this transformant. After recovery from the Tetrahymena transformant and recloning in Escherichia coli, the mutated circular plasmid again transformed Tetrahymena with stable maintenance of the circular rDNA plasmid. Transformants containing circular replicons were also obtained by using a similar plasmid from which the repeated promoter, but not the repeated replication origin, had been removed by BAL-31 deletion. We therefore propose that repeated rRNA promoters are deleterious in vivo in Tetrahymena, which normally lacks them. Transformants were obtained in 2-5 days compared with the 7-14 days required for transformation with unmutated rDNA plasmids by recombination. Similar results were obtained when a 550-base-pair segment containing the telomerase RNA gene of T. thermophila was inserted in the polylinker of the plasmid. We suggest that this plasmid is a useful vector system for transformation of Tetrahymena.

We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila. The gene encodes an RNA molecule which is 1753 nucleotides in length. The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs. We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin. In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure. Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function.

A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes. This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules. Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length. The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion. Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion. The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.

We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines.

The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region. Proceeding from the end of the rDNA molecule, the telomeric repeated sequence, (CCCCAA)n, was packaged in a non-nucleosomal complex adjacent to three phased nucleosomes. This nucleosomal structure was disrupted at the Type V repeat region, which, compared with the neighboring nucleosomal region, was more accessible to nucleases and, from both micrococcal nuclease and DNase I digestion results, was packaged in chromatin differently from the sequences flanking it on both sides. The region between the Type V repeats and adjacent to the transcription termination site was in yet another distinguishable chromatin structure as judged by its sensitivity to nucleases. It includes sites protected in chromatin and sites which were cleaved in chromatin but not detectably digested in DNA controls, suggesting that specific proteins are also associated with this region.

Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats. We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication. For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone. This type of variation was independent of the major yeast recombination pathway. The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication. Lengths of individual telomeres within a single clone varied independently of each other. Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately. These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.